Construction of ratiometric fluorescent probe based on inverse electron-demand Diels–Alder reaction for pH measurement in living cells

Abstract To endow fluorescent functions to the inverse electron demand Diels–Alder (IEDDA) reaction products, introduction of extraneous fluorophores is usually inevitable. However, this strategy is blamed for complex construction and background fluorescence. It is desirable to construct IEDDA fluorescent products in a more convenient manner. In this work, we reported the in-situ generated green fluorescence in IEDDA reaction. The fluorescence intensity of IEDDA products RA-TZ-1 and RA-TZ-2 decreased in reduced pH values (from pH 7.0–3.0). Based on this novel property, we developed a ratiometric fluorescent probe RB–RA-TZ-2 by conjugation with another probe RB-NH 2 (Em = 584 nm) with inverse response toward pH values. By single excitation, the fluorescence intensity ratio (I 584 /I 488 ) showed a linear response (R = 0.9797) to H + in pH range of 3.5–5.0. Colocalization imaging with LysoTracker Green indicated the capacity of RB–RA-TZ-2 to detect lysosomal pH (Pearson’s coefficient 0.81). Confocal microscopy was applied to measure and image intracellular pH in living cells.